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I talk about the next-generation sequencer Roche 454 based on pyrosequencing from this slide. This is one example for metagenomic sequencing of a human gut microbiome by 454 and shows the comparison with the sanger sequencing by ABI 3730xl. A shown here, one run by 454 produced more than 1 million reads with read length of 370 bases and 433 Mb about 8 times more bases were obtained. The production rate was 5 days for the 454, which is 6 time faster than the ABI 3730xl. And, we could identify 4-5 times more genes in the metagenomic sequences produced by 454 than the previous analysis by sanger sequencing. Conversely, the cost of 454 was reduced to one tenth of the sanger sequencing. And we have no cloning process, no colony picker and no bacterial culture any more. Anyway, from this result, we can easily realize that the 454 sequencer promises much deeper sequence-based metagenomics than the previous sanger sequencing.

We have sequenced human microbomes by 454 and obtained nearly 1 million reads without any problem. But the 454 reads contained many so-called artifact reads, which have the same starting base and almost same sequence each other. In these cases, 10-20% of reads were filtered out as the artifact reads. These artifact reads have no influence on the assembly but have a big problem on the quantitative mapping analysis of 454 reads to the reference genomes. Therefore, we need to remove these artifact reads prior to the mapping analysis. Artifact reads may be generated by incorrect ratio between beads number and DNA molecule. Usually, this ratio in one emulsion is one, but when multiple beads are present in one emulsion containing one DNA molecule, each bead has the same template, which produce the same sequence in different pores in which each bead embeds.

We have done more than 50 strains so far by the sanger only or sanger + 454. HMP has also released 247 draft genomes sequenced by mainly 454 to the NCBI database. My group are releasing only finished data to the public domain, simply because high quality data might be more useful for the researchers.

Sequencing of individual human microbes provides the reference genomes which is very useuful to deeply interpret the metagenomic data. For example, we can simply, accurately and directly map metagenomic shotgun reads or genes identified in the metagenomic data to bacterial genomes to assign them to the species. Very little number of genomes of human microbes were sequenced before the HMP started, but the number is dramatically increasing after the nest-gen sequencers are commercialized. Our group also working on the sequencing of human microbes particularly microbes isolated from the Japanese.

2009 hattori metagenomics

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Metagenomics: a gene-centric approach for the human gut microbiome research Masahira HATTORI Center for Omics and Bioinformatics / Dept. of Computational Biology Graduate School of Frontier Sciences, University of Tokyo http://www.cb.k.u-tokyo.ac.jp/hattorilab ・ Human-associated pathogens and commensals including intestinal microbiota ・ Symbionts in insects ・ Host-microbial interactions The Kashiwa campus

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Enriched COGs only in microbiome H Comparative metagenomics between different microbiomes is powerful to identify enriched or depleted genes in an individual microbiome Frequency H High G B C D E F A COG Commonly enriched COGs among all microbiomes Various environmental microbiomes Depleted COGs in microbiome A Low

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The adult- and infant-types Weaning may be the time to change from the infant type to the adult-type. No strong association was found within family samples Adult-type ： stable, robust to environment Infant-type ： unstable, sensitive to environment Overall sequence similarity of genes between individual microbiomes by reciprocal pairwise blastp analyses Adults/ children Americans Unweaned infants Soil Sea Whale fall ・ Relatively high similarity among adults and weaned children ・ Relatively high variation among unweaned infants The gut microbiota may be unique to individual

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Taxonomic analysis of the Japanese gut microbiota based on mapping of metagenomic reads (Phylum level) Actinobacteria Bacteroidetes Firmicutes

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The genomes of 27 Bacteroides species have been sequenced. Bacterial composition at the species level in the same genus by mapping of metagenomic reads

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Bacterial composition at the species level in the Bifidobacteria The genomes of 14 Bifidobacteriaum species have been sequenced.

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The microbial composition is highly varied but the functionality is uniform between individuals. Turnbaugh PJ et al. Nature 2009

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Conclusion 1. The functionality of gut microbiome is largely affected by diet. 2. Intestinal microbes may have evolved to acquire and accumulate functions advantageous for colonization of gut habitat, while eliminating undesired appendages that could result in sensing for pro-inflammatory responses, towards maintenance of host homeostasis. 3. Many function-unknown genes are conserved and are present in intestinal microbes. 4. The microbial diversity is highly varied but the functionality is similar between individuals 5. The gut microbiota may be unique to individual and the origin of intestinal microbiota is unknown . (No strong association of the microbiota was found within the family)

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Next and ongoing plans <ul><li>More sampling of Japanese healthy individuals. </li></ul><ul><li>・ To standardize Japanese intestinal microbiome (including probiotics-treated subjects and long-term chase of the same subjects) </li></ul><ul><li>2) Sampling of disease-afflicted subjects (IBD, colon cancer, allergy…..) and comparison with samples of healthy subjects </li></ul><ul><li>・ To identify bacteria, genes and gene products associated with the pathogenecity of the disease. </li></ul><ul><li>Comparison between samples of different nations who have different dietary style and genetic background each other. </li></ul><ul><li>・ To know how diet and genetic background affect the intestinal microbiota </li></ul>○ Sequencing of individual microbes isolated from human body sites More than 100 strains isolated from the Japanese ○ Metagenomics and 16S analysis of human intestinal microbiomes. ○ Functional studies of intestinal microbes using germ-free mice These works are being conducted as part of the International Human Microbiome Project that was launched in 2008.